Screening for point mutations, small base pair deletions or insertions in genes causing diseases has lately gained growing interest especially considering the development of certain diseases among an ethnic group or the predisposition of an individual patient. A variety of mutation scanning techniques (Beaudet et al., 1995, The Metabolic and Molecular Bases of Inherited Disease, Vol. 1, McGraw-Hill, Inc., New York) with mutation detection rates up to and above 90% (Guo et al., Nature Biotechnology, 15: 331-335, (1997)) have been developed over the last decade.
However, even more of interest than the knowledge about the presence of a specific mutation in a nucleic acid sequence would be the knowledge about the amount of such mutated nucleic acid sequences.
The standard technique to quantify nucleic acid sequences with mutations is a notoriously laborious combination of a polymerase chain reaction (PCR), a labeled (e.g., radioactive) restriction enzyme digestion and phospho-imager quantification (Saperstein and Nickerson, Biotechniques 10(4): 488-489(1991)). Accordingly, the sequences to quantify are amplified in a PCR, the amplified products are incubated with restriction enzymes and radioactive labeled. Only the nucleic acid sequences that carry a specific mutation, thereby comprising an additional restriction enzyme recognition site, can be cleaved with the restriction enzyme. Such a restriction enzyme digest results in uncleaved amplification products, not comprising the mutation, and cleaved amplification products, because of the mutation. Depending on their size, these products are separated by gel electrophoresis separation. This gel or a membrane after southern blotting this gel is exposed to screen sensitive to radioactivity. Since all fragments are equally labeled by radioactivity, the stimulation, which correlates with the amount of amplification product and which is caused by the radioactive disintegration, can be detected. Thus, it is possible to quantify said stimulation and the amount of the different amplification products, respectively, with a phospho-imager.
Beside the time consuming setup, the limits of the detection range for quantification are reached if less than 5% of the allelic sequences of the genome carry a mutation. In other words, if only 1% or less of the nucleic acid sequences carries a mutation, this mutation is not detectable and, thus, cannot be quantified.
Additionally, not every mutation can be detected by this technique, because restriction enzymes that are able to distinguish between sequences with or without mutation are often not available.
Recently, considering the fact that PCR technology has been proven to be a powerful tool for nucleic acid analysis, a highly elaborate quantification assay for PCR products, the so called TaqMan(trademark) PCR (PE Applied Biosystems, Germany), was suggested (Livak et al., PCR Methods Applic. 4:357-362(1995)).
The TaqMan(trademark) technology is based on PCR wherein an additional specific, internal, fluorogenically labeled oligonucleotide probe is used. Typical amplicons range in size from 100 bp to 1200 bp. The probe specifically anneals between the forward (5xe2x80x2) and reverse (3xe2x80x2) PCR primer binding sites (the Figure). It consists of an oligonucleotide with a 5xe2x80x2-reporter dye (e.g., FAM, 6-carboxyfluorescein) and a quencher dye (e.g., TAMRA, 6-carboxytetramethylrhodamine) which compensates the emission spectra of the reporter dye as long as both dyes are attached to the probe. While performing the standard PCR, the 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity of conventional thermostable Taq polymerase is exploited, which degrades the internal fluorogenic probe thereby releasing the fluorogenic signal of the 5xe2x80x2-reporter dye. Thus, the fluorescence signals are detectable and can be quantified. An ABI PRISM(trademark) Sequence Detection System (PE Applied Biosystems, Germany) is normally used to detect the fluorescent signals. An improvement of the TaqMan(trademark) technology was the introduction of, instead of an endpoint measurement, the measurement of the released fluorescent emission continuously during the PCR amplification (Heid et al., Genome Research, 6:, 986-994(1996)). Since the exponential accumulation of the fluorescent signal directly reflects the exponential accumulation of the PCR amplification product, this reaction is monitored in real time. From the output data of a so-called Real Time PCR, quantification a reliable back calculation to the input target DNA sequence is possible.
For the detection of minimal alterations on nucleic acid sequences found within alleles, genetic polymorphisms or the resulting transcripts the TaqMan(trademark) technology can be adjusted. In this case, for every allele or polymorphism to be detected by TaqMan(trademark) technology a specific oligonucleotide probe must be designed and used. These oligonucleotide probes differ in one or more nucleotides in accordance with the allele the probe shall bind to for TaqMan(trademark) PCR assay. Additionally, for every allele the corresponding oligonucleotide probes comprise a different colored fluorescent reporter dye. Thus, by performing the TaqMan(trademark) PCR assay every allele or polymorphism is recognized by the probe that specifically binds to its sequence. During PCR, the fluorogenic signal of the specific probe is released. Since the released fluorogenic signals are different for every allele, it can be determined how many different alleles are present. However, to determine the quantity of each different allele, this method is unreliable or even not applicable.
Assuming that alleles only differ in one base, the corresponding fluorogenic probes used for TaqMan(trademark) PCR assay also differ in only one base. In this case the specificity of the quantification assay is due to an inhibition of hybridization. Accordingly, the fluorogenic probes are of minor specificity. Since a probe specific for one allele differs from another allele only in one base, this minor difference will not prevent binding of said probe to the otherxe2x80x94unspecificxe2x80x94allele. Thus, it happens that in a TaqMan(trademark) PCR assay the same fluorogenic signal will be released from different alleles. Accordingly, this fluorogenic signal cannot be used for a reliable quantification of the corresponding allele.
To summarize, technologies as known from the prior art are not suitable to quantify alleles or nucleic acid sequences with point mutations, small base pair deletions or insertions, especially, when said sequences represent less than 10% of the allelic sequences in the genome.
It is, thus, an object of the present invention to provide a highly sensitive and reliable assay capable to distinguish and especially to quantify nucleic acid sequences that differ in at least one base.
To achieve the foregoing and other objects, the present invention inter alia comprises the following, alone or in combination:
A method of quantifying alleles comprising nucleic acid sequences differing in at least one base wherein an allele-specific primer, forming a match with one allele but a mismatch with the other(s), is used for a real time PCR resulting in amplification of the allele that forms a match with the primer followed by quantification of said amplified allele as well as of the non-amplified allele;
the method as above, wherein said non-amplified allele forms a mismatch with the terminal 3xe2x80x2 nucleotide of the allele-specific primer;
the method as above, wherein said allele-specific primer comprises additionally at least one mismatch;
the method as above, wherein said additional mismatch is at position xe2x88x922 to xe2x88x9210 of the 3xe2x80x2 tail of said allele-specific primer;
the method as any above, wherein said allele-specific primer forms a match with a minor allele present in smaller amounts than the non-amplified allele(s);
allele-specific primers with the sequence:
AS5 (SEQ ID No.: 1);
AS6 (SEQ ID No.: 2);
AS7 (SEQ ID No.: 3);
PIRA1 (SEQ ID No.: 5),
AS1 (SEQ ID No.: 11),
AS2 (SEQ ID No.: 12),
AS3 (SEQ ID No.: 13), or
AS4 (SEQ ID No.: 14);
allele-specific primers as above used for a method of quantification of alleles comprising mitochondrial DNA sequences differing in at least one base.